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MB Sample ID: SA206941
Local Sample ID: | 190905_19_0009_UCLM_CCM_042 |
Subject ID: | SU002243 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male |
Cell Biosource Or Supplier: | CLS |
Cell Strain Details: | In vitro spontaneously transformed keratinocytes from histologically normal skin |
Cell Primary Immortalized: | HaCaT |
Cell Passage Number: | Up to the 15th passage |
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Sample Preparation:
Sampleprep ID: | SP002249 |
Sampleprep Summary: | Keratinocytes were defrosted on ice and proteins were precipitated from the lysed cell samples by adding the extraction solvent spiked with metabolites not detected in unspiked cell extracts. These metabolites, considered as internal standards, were tryptophan-d5, Anthranilic acid-(ring-13C6), Phenylthiohydantoin (PTH)-valine, Glycocholic-2,2,4,4-d4 acid (Sigma Aldrich). Then, cell extracts were incubated at -20 ˚C for 1 hour and after that, samples were vortexed and centrifuged at 18,000 x g for 10 minutes at 4 ºC. Supernatants were collected and kept on ice. A second extraction was performed from the remaining pellets, following the same steps described above. Supernatants obtained from the second extraction were collected and put together with the supernatants of the first extraction. Finally, these supernatants were dried under vacuum, reconstituted in water, resuspended with agitation for 10 minutes, centrifuged at 18,000 x g for 5 minutes at 4ºC, and transferred to vials for UHPLC-MS analysis. |